猪波形蛋白对PRRSV感染Marc-145细胞的抑制作用

Role of Swine Vimentin in PRRSV Infection to Marc-145 Cells

为了研究波形蛋白在介导猪繁殖与呼吸综合征病毒(PRRSV)感染细胞过程中的作用,利用RT-PCR方法从PRRSV非易感细胞系猪肾细胞系PK-15细胞中扩增目的基因,克隆入pET-28a(+)载体,转化大肠杆菌BL21(DE3)中进行诱导表达。表达的重组猪波形蛋白经SDS-PAGE和Western blot鉴定和纯化后免疫新西兰大白兔制备多克隆抗体。利用病毒抑制试验检测重组猪波形蛋白及多克隆抗体在PRRSV感染Marc-145细胞过程中的作用。结果表明,成功扩增猪波形蛋白基因并克隆入pET-28a载体,经诱导后得到高效表达,纯化后免疫兔子产生高价血清抗体(105)。病毒阻断结果表明,猪波形蛋白及多克隆抗体均能阻断PRRSV感染Marc-145细胞。这为以波形蛋白为基础的PRRSV受体阻断抑制剂的研究提供了实验依据。

To investigate the function of swine vimentin in PRRSV infection, the swine vimentin gene from a PRRSV non - susceptible cell lines PK - 15 cells was amplified by RT - PCR and cloned into pET - 28a vector. The recombinant swine vimentin protein was expressed in Escherichia coli BL21 （ DE3 ） and was i- dentified by SDS- PAGE and Western blot. After purification, the target protein was used to immunize New Zealand rabbit to produce polyclonal antibodies. The recombinant swine vimentin protein and polyclonal anti- bodies were used to test blocking function of PRRSV infection to Marc - 145 cells by virus inhibition. The re- sults showed that the swine vimentin gene was amplified successfully from PK - 15 cells and cloned into pET - 28a vector. The rabbit anti - vimentin antibodies were produced with the titer of 1 × 10^5. PRRSV infection to Marc - 145 cells was blocked partially by the recombinant vimentin protein while blocked completely by the an- tibodies. These results provide additional information for the research of PRRSV inhibitor based on vimentin.